川芎嗪对MPTP诱导大鼠中脑多巴胺神经元损伤所致帕金森病的脑神经保护机制研究

发布时间：2018-06-16 02:50

本文选题：川芎嗪 + 帕金森氏病　；参考：《第四军医大学》2015年硕士论文

【摘要】：目的:帕金森病(Parkinson’S disease PD)是一种较为常见的椎体外系变性性疾病,由于其确切的发病机制迄今尚不十分明确,仍没有根治的办法。临床上多选用药物治疗延缓病情恶化,但由于长期用药导致疗效降低、毒副作用不耐受等,使患者不得不减少剂量或终止药物治疗。因此,寻找治疗效果好、安全性高的PD药物是当前的研究热点。川芎嗪(Tetramethylpyrazine,TMP)是从中药川芎中提取的有效成分,其被广泛应用于治疗神经和心血管性疾病。近年来,一些研究已经表明,川芎嗪在体内外具有神经保护作用。川芎嗪也被发现具有缺血性脑损伤的保护作用,并可促进细胞增殖与分化。1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)是一种神经毒素,对参与PD发生的神经元具有靶向作用。因此,MPTP已被广泛应用于帕金森病动物模型的建立。本实验的目的是探讨川芎嗪对MPTP所致大鼠中脑多巴胺神经元损伤的保护作用及可能的抗凋亡抗氧化机制,为帕金森氏病的临床治疗提供分子基础。方法:1.动物分组与模型处理大鼠随机分为4组,每组10只,分别为:对照组(Con,生理盐水注射),MPTP组(MPTP与生理盐水混合注射),TMP组(TMP注射),TMP+MPTP组(TMP与MPTP混合注射)。TMP(TMP 20mg/kg/d,TMP溶解于3%DMSO中,腹腔注射)给药7天。在第7天,大鼠黑质(SN)注入MPTP(1μmol MPTP溶解于2μl生理盐水中,参照坐标:AP:-5.0 mm,ML:±2.1 mm,DV:-7.7 mm,使用30量规针头从大鼠前卤,中线和头骨表面以0.35μl/min的速度注入),随后继续TMP给药7天。步行阶梯实验在第14天的14:00-18:00经行。实验后,将大鼠处死并解剖,取出纹状体和黑质以备后续实验。2.步行阶梯实验步行阶梯实验用于评价大鼠前后肢体的协调能力。在测试前大鼠被训练3天进行横跨梯子的训练,记录第14天进行的3次测试的平均成绩。3.多巴胺及其代谢产物的测定大鼠脱颈髓处死,冰台上迅速断头将纹状体取出,称重,于-80℃储存。检测时加入含有DHBA内标的0.1mol/L的高氯酸溶液0.5ml,匀浆,于4℃低温离心,15000r/min,离心l0min,取上清50μl用于高效液相色谱电化学检测。4.TBARS和GSH的测定参照说明书方法测定巴比土酸活性物质(TBARS)和谷胱甘肽(GSH)含量5.免疫组化染色将处理后的大鼠脑组织-20℃冷冻并连续切片(5um),置于玻璃载玻片上,在4oC下细胞与特异性抗体共同孵育过夜,与Cy3抗体共同培养。然后置于共聚焦显微镜下观察拍照。6.免疫组化检测酪氨酸羟化酶(TH)阳性细胞数造模一周后大鼠断头处死,在冰上取中脑腹侧,立即将其置于4%多聚甲醛中固定,蔗糖脱水后进行冰冻切片,以15μm厚度连续切片,晾片后加大鼠抗TH单克隆一抗(1:1000),37℃下孵育l小时,PBS洗3次每次5分钟,加入荧光标记的兔抗大鼠二抗,37℃孵育60分钟,PBS洗3次,每次5分钟经封皮后荧光显微镜镜下观察黑质致密部(SNc)的TH阳性细胞数,图像分析系统计数,每组取5个动物,每个动物取5张切片,计算平均数值。7.Western Blot实验检测相关蛋白表达水平结果:1.川芎嗪对MPTP诱导的运动障碍的影响与Con组大鼠相比,MPTP组大鼠明显花费更多时间通过阶梯,证实MPTP诱导大鼠产生运动障碍;与MPTP组相比,TMP+MPTP组大鼠通过阶梯时间明显缩短,结果表明,TMP的干预,使MPTP诱导的大鼠运动障碍得到改善。2.川芎嗪对MPTP诱导的DA能神经元损伤的影响与Con组相比,MPTP组大鼠酪氨酸羟化酶免疫反应明显减少,DA和DOPAC水平明显降低;应用TMP干预后,抑制了DA和DOPAC水平的降低,可显著防止MPTP诱导的DA能神经元损伤。3.TMP对MPTP诱导的细胞凋亡的影响为了评估细胞凋亡的相关改变,我们检测Bax和Bcl-2的蛋白表达。在黑质中,MPTP组大鼠Bax蛋白表达增加,Bcl-2蛋白表达降低。而MPTP诱导的Bax和Bcl-2的蛋白表达改变能被TMP有效抑制。另外,我们检测了细胞质中细胞色素c的释放,与Con组相比,MPTP组细胞色素c释放增多,而TMP干预后细胞色素c的释放得到有效抑制。我们同时也检测了capase3的剪切,MPTP可促进capase3发生剪切,而TMP可显著降低capase3的剪切。这些结果说明TMP可抑制MPTP诱导的大鼠大脑黑质细胞凋亡。4.TMP对MPTP诱导的诱导的氧化应激的影响我们检测了TMP和MPTP对大鼠大脑黑质氧化还原平衡的影响。与Con组相比,MPTP组TBARS水平显著增加;说明MPTP引起了氧化损伤的发生。而TMP干预后可有效抑制TBARS水平的增高。MPTP组与Con组相比,GSH含量显著降低。而TMP干预后可以有效抑制其降低。与Con组相比,MPTP组Nrf2和GCLc表达明显降低降低。而TMP干预后能有效抑制Nrf2和GCLc的表达降低。这些结果说明TMP可抑制MPTP诱导的大鼠大脑黑质氧化应激损伤。结论:1.川芎嗪可以改善MPTP诱导的大鼠运动障碍并可显著防止MPTP诱导的多巴胺能神经元损伤。2.川芎嗪的抗凋亡,抗氧化作用是其产生保护神经元损伤作用的可能机制。3.川芎嗪的抗凋亡作用是通过其抑制Bax表达,增加Bcl-2表达,抑制细胞色素c的释放,抑制caspase3的裂解来实现的;其抗氧化作用与调节Nrf2和GCLc有关。
[Abstract]:Objective: Parkinson's disease (Parkinson 'S disease PD) is a more common degenerative disease of the vertebral body. Because its exact pathogenesis is not yet very clear, there is still no cure for it. The people have to reduce the dose or terminate the drug treatment. Therefore, to find the effective and safe PD drugs is the current research hotspot. Ligustrazine (Tetramethylpyrazine, TMP) is an effective component extracted from Ligusticum chuanxiong, which is widely used in the treatment of nerve and heart blood tube diseases. In recent years, some studies have shown that Ligustrazine. Tetramethylpyrazine has also been found in the body and in vivo. Ligustrazine has also been found to have protective effects on ischemic brain damage, and can promote cell proliferation and differentiation,.1- methyl -4- phenyl -1,2,3,6- four hydropyridine (MPTP) is a neurotoxin that is targeted to neurons involved in PD. Therefore, MPTP has been widely used in the animal model of Parkinson's disease. The purpose of this experiment was to explore the protective effect of Ligustrazine on the injury of dopamine neurons in the mesencephalon of MPTP rats and the possible anti apoptosis antioxidation mechanism, and to provide the molecular basis for the clinical treatment of Parkinson's disease. Methods: 1. animal groups and model rats were randomly divided into 4 groups, 10 in each group, Con, Saline injection), group MPTP (MPTP and saline mixed injection), group TMP (TMP injection), TMP+MPTP group (TMP and MPTP mixed injection).TMP (TMP 20mg/kg/d, TMP dissolved in 3%DMSO, intraperitoneal) for 7 days. On the seventh day, the rat substantia nigra was injected into 2 mu saline. 7 mm, using the 30 gauge needle from the anterior brine of the rat, the middle line and the surface of the skull at the speed of 0.35 mu l/min, and then continue to TMP for 7 days. The walking ladder experiment was performed on the fourteenth day of 14:00-18:00. After the experiment, the rats were killed and dissected, the striatum and the substantia nigra were taken out for the follow-up test of the walking ladder experiment of the.2. walking ladder test used for evaluation. The rat's ability to coordinate the limbs before and after the test. The rats were trained for 3 days before the test. The average score of the 3 tests on fourteenth days was recorded,.3. dopamine and its metabolites were measured. The rats were removed from the cervix, and the striatum was removed, weighed and stored at -80 C. The DHBA internal standard was added to the test. 0.1mol/L's perchloric acid solution 0.5ml, homogenate, centrifugation at 4 centigrade, 15000r/min, centrifugation l0min, and supernatant 50 mu L for high performance liquid chromatography electrochemical detection.4.TBARS and GSH determination reference manual method for determination of barbiulic acid active substance (TBARS) and glutathione (GSH) content 5. immuno histochemical staining of brain tissue at -20 C after treatment Frozen and continuous slice (5um), placed on glass slides, incubated with specific antibodies in 4oC cells for the night, co culture with Cy3 antibody, and then placed under confocal microscope and observed and photographed.6. immunohistochemistry for the detection of tyrosine hydroxylase (TH) positive cells for a week, the rats were killed and the midbrain was taken on the ice, immediately on the ventral side of the brain. It was fixed in 4% polyformaldehyde. After the sucrose was dehydrated, the frozen section was sliced and sliced with 15 m thickness. After airing, the rat anti TH monoclonal antibody (1:1000) was increased, l hours were incubated at 37 degrees, PBS was washed for 3 times for 5 minutes. The rabbits were added to the fluorescent label for two anti rats, 37 centigrade was incubated for 60 minutes, the PBS was washed for 3 times, and the fluorescence microscopy after 5 minutes after 5 minutes was used. The number of TH positive cells in the dense part of the substantia nigra (SNc) was observed under the microscope, and the image analysis system was counted. 5 animals were taken in each group. 5 slices of each animal were taken, and the average value of.7.Western Blot was calculated to determine the expression level of related proteins: 1. the effect of Ligustrazine on the obstacle induced by MPTP was compared with that of the Con group, and the rats in the MPTP group were significantly spent. More time passed the ladder to confirm that MPTP induced the dyskinesia in rats; compared with the MPTP group, the rats in the group TMP+MPTP were significantly shortened through the ladder time. The results showed that the intervention of TMP, caused by MPTP induced dyskinesia, could improve the effect of.2. tetramethylpyrazine on the DA energy damage induced by MPTP, compared with the Con group, and the MPTP group rat tyrosine. The immunoreaction of hydroxylase was significantly reduced and the levels of DA and DOPAC were significantly reduced. The reduction of DA and DOPAC levels was inhibited by the use of TMP stem. The effects of MPTP induced DA neurons on the apoptosis induced by MPTP induced by.3.TMP were obviously prevented. We detected the protein expression of Bax and Bcl-2. In the substantia nigra, we detected the protein expression of Bax and Bcl-2 in the substantia nigra. The expression of Bax protein in the MPTP group increased and the expression of Bcl-2 protein decreased. The protein expression changes of Bax and Bcl-2 induced by MPTP could be effectively suppressed by TMP. In addition, we detected the release of cytochrome C in the cytoplasm. Compared with the Con group, the c release of cytochrome in the MPTP group increased, and the release of cytochrome MPTP was effectively suppressed after the TMP intervention. At the same time, the shear of capase3 was also detected, and MPTP could promote the shear of capase3, while TMP significantly reduced the shear of capase3. These results suggest that TMP can inhibit the effect of MPTP induced rat cerebral nigra apoptosis.4.TMP on the induced oxidative stress induced by MPTP, and we detected the redox balance of TMP and MPTP on rat brain black matter. Effect. Compared with group Con, the level of TBARS in group MPTP was significantly increased, indicating that MPTP caused oxidative damage, and TMP intervention could effectively inhibit the level of TBARS,.MPTP group was significantly lower in GSH content compared with Con group, while TMP intervention could effectively inhibit its decrease. The intervention can effectively inhibit the decrease of Nrf2 and GCLc expression. These results suggest that TMP can inhibit the oxidative stress injury in rat cerebral substantia nigra induced by MPTP. Conclusion: 1. Ligustrazine can improve the dyskinesia induced by MPTP and prevent the MPTP induced dopaminergic neurons to damage the apoptosis of.2. tetramethylpyrazine, and the antioxidant effect is its production. The possible mechanism for the protection of neuron damage,.3. tetramethylpyrazine's anti apoptosis effect is realized by inhibiting the expression of Bax, increasing the expression of Bcl-2, inhibiting the release of cytochrome c and inhibiting the cracking of Caspase3, and its antioxidant effect is related to the regulation of Nrf2 and GCLc.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.5

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